Disulfide interchange enzymic

There are at least two types of enzyme systems involved in the formation and breakage of disulfide bonds of cystine residues in proteins. A thiol-disulfide interchange enzyme (protein disulfide-isomerase EC 5.3.4.1 other name, S-S-rearrangase) was first described in 1963 ( 47) and was subsequently purified from beef liver (48,49). The molecular weight of the enzyme is 42,000. The enzyme contains three half-cystine residues, one of which must be cysteine in order for the enzyme to be active. The enzyme catalyzed the rearrangement of random incorrect pairs of half-cystine residues to the native disulfide bonds in several protein substrates. Low levels of mercaptoethanol were required for activity unless the enzyme was reduced prior to use. The efficiency of the enzyme in catalyzing the interconversion of disulfide bonds was found to be a function of the number of disulfide bonds in the substrate. Purification of a thiol-disulfide interchange enzyme from Candida claussenii has been described recently (50). 

The reaction catalyzed by the thiol-disulfide interchange enzyme may be depicted as shown in Equation 1 ... 

In contrast, when the protein is allowed to form the proper tertiary structure before disulfide formation, essentially all the enzymatic activity is recovered. The disulfide interchange enzyme acts on newly made proteins, catalyzing the breakage and rejoining of disulfides in a protein. Combined with the action of the chaperonins, the enzyme helps the protein achieve its final, native state, with all the disulfides formed appropriately. 

This enzyme [EC 5.3.4.1], also known as S—S rearran-gase, catalyzes the rearrangement of intrachain or interchain disulfide bonds within proteins to form then-native structures. The enzyme requires reducing agents or the partly reduced enzyme. The reaction operates by sulfhydryl-disulfide interchange. 

In an earlier experiment, Jori et al. (14) reported that methionyl residues are important in maintaining the tertiary structure of lysozyme. The introduction of a polar center into the aliphatic side chain of methionine, as a consequence of the conversion of the thioether function to the sulfoxide, may bring about a structural change of the lysozyme molecule which, in turn, reduces the catalytic efficiency. When ozonized lysozyme was treated with 2-mercaptoethanol in an aqueous solution according to the procedure of Jori e al. (14), the enzyme did not show any increase in its activity. This may be explained in two ways. In one, such reactions are complicated by many side reactions, e.g. sulfhydryl-disulfide interchange, aggregation and precipitation of the modified enzyme (24-26). In the other, the failure to recover the activity of the enzyme may by associated with the extensive oxidation of other residues. 

A rearranging enzyme has been found in microsomes which is a general catalyst for disulfide interchange reactions required in the reoxidation process (203-205). This enzyme may be very important biologically but is used here solely as a reagent for the study of RNase and will not be considered in detail. 

Glutathione helps to maintain the sulfhydryl groups of proteins in a reduced state. An enzyme, protein-disulfide reductase, catalyzes sulfhydryl disulfide interchanges between glutathione and proteins. The reductase is important in insulin breakdown and may catalyze the reassortment of disulfide bonds during polypeptide chain folding. 

Further structure-activity relationship (S AR) analyses of other cytoprotective enzyme inducers revealed the fact that all inducers can react with thiol/disulfide groups by alkylation, oxidoreduction, or thiol-disulfide interchange [Dinkova-Kostova and Talalay, 1999]. In fact, the capability of enzyme inducers to induce cytoprotective enzymes is well correlated with their reactivity with thiols. These results suggested a cellular sensor of inducers with highly reactive sulfhydryl groups, possibly reactive thiols in cysteine residues of a sensor protein. Nevertheless, the initial search for the sensor protein by using radioactively labeled inducers was not successful due to the abundance of thiol groups presented in many proteins in cells [Holtzclaw et al., 2004]. The molecular mechanism by which cytoprotective enzymes are induced remained to be elucidated. 

In a classic study on bovine pancreatic ribonuclease A at 90°C and pH conditions relevant for catalysis, irreversible deactivation behavior was found to be a function of pH (Zale, 1986) at pH 4, enzyme inactivation is caused mainly by hydrolysis of peptide bonds at aspartic acid residues as well as deamidation of asparagine and/or glutamine residues, whereas at pH 6-8, enzyme inactivation is caused mainly by thiol-disulfide interchange but also by fi-elimination of cystine residues, and deamidation of asparagine and/or glutamine residues. 

The pursuit of the tertiary protein structural problem led Anfinsen to the discovery of a microsomal enzyme that catalyzes sulfhydryl-disulfide interchange and accelerates the refolding of denatured proteins which contain disulfide bonds in vitro. The kinetics of this folding accounts for the rate of folding of newly synthesized proteins in vivo. It was shown, however, that the renaturation required very dilute solutions in many cases to avoid aggregation of the protein in place of proper folding. 

It has also been possible to modulate enzyme function (29) by introduction of a disulfide bridge spanning the active site cleft of T4 lysozyme (Fig. 1). In order to avoid possible thiol-disulfide interchange with Cys-S4 and Cys-97 in the native structure, these two residues were converted to threonine and alanine, respectively, with no loss in the activity or stability of the enzyme. The latter protein was then further modified by replacing Thr-21 and Thr-42 by cysteines that spontaneously oxidized to the desired disulfide. This oxidized enzyme form exhibited no detectable activity, although some activity (7% of wild type) was restored on reduction of the linkage. This represents a novel use of the disulfide bond to modulate catalytic activity. 

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